牛细小病毒SYBR GreenⅠ实时荧光定量PCR检测方法的建立

Establishment of SYBR GreenⅠ real-time q PCR for the detection of bovine parvovirus

为建立检测牛细小病毒的SYBR GreenⅠ荧光定量PCR方法,本研究通过提取牛细小病毒(ATCC VR-767)核酸,扩增NS1基因片段(大小约为171 bp),将NS1基因片段克隆到pMD19-T载体中,并将重组质粒p MD19-T-NS1/DH5α作为标准样品,用于建立SYBR GreenⅠ实时荧光定量PCR检测方法。研究结果显示,扩增基因片段长约171 bp,符合目的基因大小,经鉴定所构建的重组质粒正确。用该质粒测得SYBR GreenⅠ实时荧光定量PCR的最低检测量为53.3 copies/uL。用该方法检测牛细小病毒、牛轮状病毒、牛病毒性腹泻病毒、牛呼吸道合胞体病毒和猪细小病毒,结果显示,只有牛细小病毒可以被检测到,而其他病毒均未被检出,表明该方法具有良好的特异性。重复性试验结果表明,批内和批间重复变异系数均小于1%,表明该方法具有良好的重复性。结果表明,本研究建立的基于NS1基因的牛细小病毒SYBR GreenⅠ实时荧光定量PCR检测方法具有良好的特异性、灵敏性、重复性,可用于牛细小病毒感染的检测。

In order to establish a SYBR GreenⅠreal-time fluorescent quantitative PCR assay to detect bovine parvovirus(BPV),in this study we extracted the nucleic acid of BPV(ATCC VR-767)and then amplified NS1 gene fragment(171 bp in size).The NS1 gene fragment was cloned into pMD19-T vector,and the recombinant plasmid p MD19-T-NS1/DH5αwas used as the standard sample to establish a SYBR GreenⅠreal-time fluorescent quantitative PCR assay.In result,the length of amplified gene fragment was 171 bp,which was consistent with the size of target gene.The recombinant plasmid constructed was identified to be correct.The minimum detectable amount of the SYBR GreenⅠreal-time PCR was 53.3 copies/u L.This method was used to detect bovine parvovirus,bovine rotavirus,bovine viral diarrhea virus,bovine respiratory syncytial virus and porcine parvovirus.In result,only bovine parvovirus could be detected and the other viruses could not be detected,which indicated that the method had good specificity.The results of repeatability test showed that the variation coefficients of intra-assay and inter-assay were both less than 1%,which indicated that the method had good repeatability.In summary,the SYBR GreenⅠreal-time quantitative PCR assay based on NS1 gene has good specificity,sensitivity and repeatability,and can be used for the detection of bovine parvovirus infection.